Protein Scaffold-Activated Protein Trans-Splicing in Mammalian Cells

• Published in: Journal of the American Chemical Society Vol. 135; no. 20; pp. 7713 - 7719
• Main Authors: Selgrade, Daniel F, Lohmueller, Jason J, Lienert, Florian, Silver, Pamela A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.05.2013
• Subjects: Cells, Cultured; Enzyme Activation; Exteins; Humans; Inteins; luciferase; Luciferases - chemistry; Luciferases - metabolism; mammals; Protein Splicing; Proteins - chemistry; Proteins - metabolism; scaffolding proteins; trans-splicing
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/ja401689b

Conditional protein splicing is a powerful biotechnological tool that can be used to rapidly and post-translationally control the activity of a given protein. Here we demonstrate a novel conditional splicing system in which a genetically encoded protein scaffold induces the splicing and activation of an enzyme in mammalian cells. In this system the protein scaffold binds to two inactive split intein/enzyme extein protein fragments leading to intein fragment complementation, splicing, and activation of the firefly luciferase enzyme. We first demonstrate the ability of antiparallel coiled-coils (CCs) to mediate splicing between two intein fragments, effectively creating two new split inteins. We then generate and test two versions of the scaffold-induced splicing system using two pairs of CCs. Finally, we optimize the linker lengths of the proteins in the system and demonstrate 13-fold activation of luciferase by the scaffold compared to the activity of negative controls. Our protein scaffold-triggered conditional splicing system is an effective strategy to control enzyme activity using a protein input, enabling enhanced genetic control over protein splicing and the potential creation of splicing-based protein sensors and autoregulatory systems.